Influenza viruses are pathogens that cause viral respiratory disease, and are largely classified into types A, B and C based on the antigenic differences in their NP (nucleocapsid) and M (matrix) proteins. Among them, type A is further classified into subtypes H1 to H15 based on the antigenicity of their HA (hemagglutinin) proteins, and into subtypes N1 to N9 based on their antigenic characteristics of their NA proteins (see Wiely DC and Skehel TT, Ann Rev Biochem, 56:365-394, 1987). Among them, subtypes H1, H2, H3, N1 and N2 generally cause disease in humans. The H and N antigens that appear in birds generally do not cause disease in humans, but can change to types that cause disease even in humans, when genetic mutations in the viruses occur or when the viruses exchange genes with antigens that cause disease in humans. If such new influenza viruses against which humans have no prior immunity emerge, they can cause a worldwide pandemic.
In recent years, various diagnostic kits for detecting such influenza viruses have been developed. In particular, as the number of deaths caused by highly pathogenic avian influenza viruses such as swine influenza has increased rapidly, rapid diagnostic kits for early diagnosis have been required. In the case of diseases (such as a novel swine influenza pandemic in 2009) that spread to many and unspecified persons in public places such as schools and work sites, the quarantine of early infected persons upon diagnosis is required to prevent pandemic, and rapid and accurate early detection systems are required. Such systems are national public systems necessary for public safety. Rapid diagnostic systems for on-site diagnosis should be small in size so as to be easy to carry, should be simple to use, and should provide a user interface so as to be easily operated by the user.
Three main conventional methods used to diagnose influenza are virus culture test, rapid antigen test, and PCR test. Among these methods, the virus culture method that is a traditional diagnosis method is accurate, but has limitations in that it is time-consuming and complicated, and thus provides diagnostic results at a late time. Also, it cannot be applied for treatment. In addition, the rapid antigen test based on antigen-antibody reactions for primary identification of influenza-infected persons is a convenient and rapid test that requires a time of about 30 minutes, and has an advantage in that it does not require an additional system and technical manpower for the test. However, the rapid antigen test has a disadvantage in that, because it detects only the nucleoprotein of types A and B, it has low sensitivity and accuracy, and thus can cause a wrong diagnosis leading to the death of the patient. Thus, it is difficult to apply for on-site diagnosis. The diagnosis of influenza subtypes becomes an index enabling prediction of resistance to influenza treatment drugs, and indicates epidemiologically significant disease prevalence. Currently, the diagnosis of influenza subtypes is possible only by RT-PCR and is costly, and a H1/A/B diagnostic reagent (SD Co., Ltd.) is the only reagent for rapid diagnosis of antigen, which enables the identification of subtypes of type A, and other reagents have not been developed and used worldwide.
Real-time PCR that has been used as a diagnostic test for swine influenza is the most effective mean for diagnosis of infectious diseases due to its high sensitivity and accuracy. However, it requires a test time of 2-4 hours, and may not be used for on-site diagnosis. For these reasons, when performed by a professional organization, a time of about 2-3 days is generally required for confirming the test result. In addition, it is costly. Thus, this real-time PCR cannot be used as an initial countermeasure in a national scope at a time point at which an infectious disease pandemic is expected. Furthermore, avian influenza infecting various kinds of birds, including chickens, turkeys and wild birds, as well as humans, spreads rapidly with diverse pathogenicities. Thus, the infected birds are killed in most of countries in the world in case highly pathogenic avian influenza occurs. Also, a country in which the highly pathogenic avian influenza has occurred is seriously damaged, because it cannot export chicken products.
Accordingly, the present inventors have made extensive efforts to solve the above-described problems, and as a result, have developed an influenza detection kit capable of not only detecting the nucleoproteins of influenza types A/B, but also simultaneously detecting the hemagglutinins of influenza subtypes H1, H3 and H5 without cross reactions, and have found that the use of the developed influenza detection kit enables the on-site detection and diagnosis of even influenza subtypes, including avian influenza subtype H5, thereby completing the present invention.